DESCRIPTION (applicant's description): Integration of extracellular signals is an essential function of the plasma membrane. For seven transmembrane segment receptors this function is accomplished by heterotrimeric G proteins that couple receptor activation to specific effectors. While numerous receptors and effectors have been identified and the crystal structure of several G proteins has been solved, the way in which these molecules interact with membranes or participate in chemical reactions comprising the signal transduction cascade remains poorly understood. The main objective of this project is to provide insights into these processes. Total internal reflection fluorescence microscopy (TIRFM) of supported membranes will be used to investigate interactions of fluorophore-labeled G protein alpha and (3y subunits with lipid membranes, either pure or incorporating molecules with which G proteins interact, in order to understand the physical chemistry of G protein interactions in a chemically defined environment. Subunit lipid modifications, including N-terminal myristoylation and thioacylation of Gi/oalpha will be examined since these may play a primary role in the dynamics of subunit interactions with membranes. Membranes approximating the composition of detergent resistant subdomains of the plasma membrane will be examined to identify molecular features that may target G proteins specifically to these areas. Proteins that associate with G proteins, such as RGS, caveolin and a channel effector, will also be examined for their effects on subunit lateral mobility and interactions. In a complementary approach, excised patches of cell membranes expressing G protein regulated muscarinic potassium channels (KACh) will be used not only to assay the functionality of fluorescently labeled and/or chemically modified G protein subunits but also to understand channel regulation in vivo on the basis of our TIRFM results. A combination of quantitative biophysical, biochemical and physiological approaches is believed to be a unique aspect of the proposed studies that should provide realistic and complementary perspectives of G protein coupled signal transduction. Moreover, these studies should help to establish a link between structural features of molecules participating in G protein coupled signaling and the physiology of the regulatory responses. Linking structural features to function is an essential step towards understanding and ameliorating conditions stemming from compromised signal transduction processes in cell membranes.